The Cancer Stem Cell Hypothesis posits the existence of a scarce stem cell population within each tumor that is resistant to chemotherapy and is responsible for generating and maintaining the bulk tumor population. Ewing sarcoma family tumors (ESFT) typify the response to treatment expected of a tumor driven by cancer stem cells. Current therapies most likely target the stem cells' progeny (the bulk of the tumor) and spare the true cancer stem cells. Consequently, although the majority of patients achieve a remission, relapse is common. ESFT contains a characteristic translocation, t(11:22), which encodes the oncogenic transcription factor EWS-FLI1. EWS-FLI1 is thought to drive tumorigenesis, so inhibition of EWS-FLI1 should be an effective treatment strategy. Because EWS-FLI1 lacks intrinsic enzymatic activity, our approach to pharmacologic inhibition is through targeting protein-protein interactions. RNA Helicase A is a critical binding partner of EWSFLI1. We have identified a lead compound, YK-4-279, that blocks EWS-FLI1 interaction with RHA. Furthermore, our preliminary data suggest that a subpopulation within ESFT cell lines with stem-like properties still expresses EWS-FLI1. Therefore, EWS-FLI1 is expected to be a key molecular target in ESFT stem cells. We hypothesize that a drug that blocks the interaction of RHA with EWS-FLI1 will target chemotherapyresistant ESFT stem cells. These studies will provide the necessary preclinical data to justify clinical trials with a compound designed specifically to target ESFT stem cells. The primary objective of this study is to develop a targeted therapy for Ewing's sarcoma stem cells (ESSC). We are proposing an integrative, collaborative research program aimed at the identification and characterization of ESSC which will allow the development of an effective therapeutic strategy to target and eliminate these cells in patients. The first aim of the study is to identify and characterize ESSC. We will employ a variety of assays that have been proposed to identify cancer stem cells, including expression of aldehyde dehydrogenase, dye efflux, the ability to form spherical aggregates under nonadherent conditions, and the expression of characteristic genes. Gene expression profiling will be hypothesis-driven, and will be performed specifically to test the hypothesis that Ewing's sarcoma is derived from the neoplastic transformation of mesenchymal stem cells. We will then demonstrate that inhibition of the EWS-FLI1/RHA interaction is lethal to chemotherapy-resistant ESSC. The final aim of the study is to identify other key signaling pathways that are amenable to small molecule targeting. This will address the possibility that inhibition of EWS-FLI1 activity alone might be insufficient to eliminate ESSC. Simultaneous targeting of multiple signaling pathways is likely to be a substantially more effective therapeutic strategy. We anticipate that at the conclusion of the project, ESSC will have been isolated and sufficiently characterized to allow the development of targeted therapies that can be rapidly translated into clinical trials and dramatically improve the prognosis of patients with Ewing's sarcoma family tumors.